Spontaneous mutations in hlyD and tuf genes result in resistance of Dickeya solani IPO 2222 to phage ϕD5 but cause decreased bacterial fitness and virulence in planta

Lytic bacteriophages able to infect and kill Dickeya spp. can be readily isolated from virtually all Dickeya spp. containing environments, yet little is known about the selective pressure those viruses exert on their hosts. Two spontaneous D. solani IPO 2222 mutants (0.8% of all obtained mutants), DsR34 and DsR207, resistant to infection caused by lytic phage vB_Dsol_D5 (ΦD5) were identified in this study that expressed a reduced ability to macerate potato tuber tissues compared to the wild-type, phage-susceptible D. solani IPO 2222 strain. Genome sequencing revealed that genes encoding: secretion protein HlyD (in mutant DsR34) and elongation factor Tu (EF-Tu) (in mutant DsR207) were altered in these strains. These mutations impacted the DsR34 and DsR207 proteomes. Features essential for the ecological success of these mutants in a plant environment, including their ability to use various carbon and nitrogen sources, production of plant cell wall degrading enzymes, ability to form biofilms, siderophore production, swimming and swarming motility and virulence in planta were assessed. Compared to the wild-type strain, D. solani IPO 2222, mutants DsR34 and DsR207 had a reduced ability to macerate chicory leaves and to colonize and cause symptoms in growing potato plants.

Bacteria can interfere with viral infection at all stages of the phage-host interaction, including adsorption, DNA entry, replication, transcription and translation, capsid assembly, and release of progeny (daughter) phages 10 . Phage-resistant mutants often avoid viral infections through mechanisms inhibiting virus adsorption to the host surface 11 . This adsorption inhibition via spontaneous mutations in genes involved with the status of the cell surface has been best characterized at the genomic level in the model phage-host system of Escherichia coli and its phage T4 12,13 . Several other studies have addressed this process in Gram-negative bacteria and their associated viruses 14,15 . Still, the knowledge about the relationship between the ecological costs of accumulating random mutations in the host genomes to achieve phage resistance, such as survival in adverse conditions, and/ or the ability to remain virulent, is limited. The effect of phage-resistance mutations on other phenotypes of the resistant cells has not been well addressed. Finally, even though the occurrence of spontaneous phage resistance has been explored in several phage-bacterium systems, little is known about these processes taking place in plant pathogenic bacteria residing in natural and agricultural settings. This subject is of particular interest given the diversity of environmental situations that bacteria face during their lives. Indeed, studies addressing spontaneous phage resistance and its ecological consequences in the important plant pathogens belonging to Pectobacterium spp. and Dickeya spp., commonly referred to as the Soft Rot Pectobacteriaceae (SRP), are limited 16-19. SRP are an excellent model for studying spontaneous phage resistance in the environment. These bacteria are considered among the 10 most important agricultural plant pathogens and cause significant losses reaching up to 40% of crop production worldwide 20 . D. solani is an emerging plant pathogen, causing disease symptoms in various crops and nonfood plants worldwide 21 . This species was first reported in 2009 in potato 22 , and has remained an important agricultural plant pathogen in most European countries 23 as well as in several agricultural regions outside Europe [24][25][26] . The bacteria are prevalent in diverse ecological niches, including bulk soil and the rhizosphere of agricultural and natural soils, in surface and rainwater, in insects and host and non-host plants 23,27,28 .
The large local population sizes that these bacteria achieve, especially in infected plants, facilitate epidemics of phage infection. Therefore, selective pressure associated with lytic bacteriophages in these settings is expected to lead to strong selection for phage-resistant mutants. The fitness of the mutants, thus, will depend not only on their virulence to plants but also on the particular environment in which bacteria interact with viruses. As SRP bacteria are often transferred between different ecological niches during their life cycles, they are often exposed to new viral infections 29 . However, the fitness costs the spontaneous phage-resistant SRP mutants pay in natural settings remain unclear.
This investigation sought to determine the extent to which spontaneous phage resistance and loss of virulence to plants are linked. This study evaluated the extent to which spontaneous phage-resistant mutants of D. solani also exhibited other phenotypic alternations that could influence their ecological fitness compared to the wildtype, phage-susceptible strain. As a model, the study investigated the spontaneous phage resistance of mutants of IPO 2222 21 selected in the presence of lytic bacteriophage vB_Dsol_D5 (ΦD5) 30,31 .
Using this model, we addressed the hypothesis that resistance to viral infection negatively impacts bacterial phenotypes important during interactions with plants, thus lowering their fitness and decreasing competition in such environments.

Selection of D. solani phage-resistant mutants with reduced virulence in planta. A total of 250
individual spontaneous ΦD5-resistant D. solani mutants were obtained in this study. All were confirmed to be D. solani IPO 2222 based on ERIC-PCR (data not shown). Two phage-resistant mutants, DsR34 and DsR207, representing 0.8% of the total mutant population, exhibited significantly reduced maceration of potato tubers compared to the WT strain ( Supplementary Fig. 1). These two phage-resistant mutants were chosen for further analysis. The selected ΦD5-resistant D. solani mutants expressed statistically significantly reduced ability to cause maceration symptoms on chicory leaves compared with the WT strain ( Supplementary Fig. 2). In two separate experiments in which potato plants grown in potting soil were exposed to the strains with a soil drench, few plants inoculated with mutants DsR34 and DsR207 (0 to 20%) in both experiments exhibited any disease symptoms. In contrast, 80 to 90% of the plants inoculated with the wild-type strain developed typical blackleg symptoms leading even to the death of the inoculated plants in both experiments. In both experiments, no symptoms were observed in control plants inoculated only with sterile Ringer's buffer ( Supplementary Fig. 3A). In addition to the significant reduction in disease incidence in plants inoculated with the two phage-resistant mutants compared to the wild-type strain (Fig. 3), the severity of infection, was significantly reduced. Although the population size of the WT strain differed statistically between individual infected plants when measured 14 days after inoculation, the bacteria were recovered from the majority of inoculated plants at densities ranging from 10 3 to 10 4 CFU g -1 of the stem tissue ( Supplementary Fig. 3B). In contrast, DsR34 and DsR207 were seldom recovered, being found both in a much lower proportion of plants than the WT strain, and at a lower population size in the stem in those few plants in which it was recovered at all. Viable cells of DsR34 were detected in only 3 plants out of 20 inoculated (1 plant in experiment 1 and 2 plants in experiment 2). In contrast, cells of DsR207 were recovered in only 4 of 20 plants inoculated plants (2 plants in experiment 1 and 2 plants in experiment 2) expressing typical infection symptoms. The average population size of the phage-resistant mutants recovered from plants in both experiments were between 10 2 to 10 3 CFU g -1 of the stem tissue. D. solani was not detected in the stems of non-inoculated negative control plants ( Supplementary Fig. 3B). www.nature.com/scientificreports/ 2222 reference genome 32 . In mutant DsR34, a mutation in the gene encoding secretion protein HlyD (locus tag: A4U42_17720, reference sequence: ANE77881.1) conferred ΦD5 resistance. In mutant DsR207, a mutation in the gene encoding elongation factor Tu (EF-Tu, locus tag: A4U42_06860, reference sequence: ANE75072.1) was responsible for phage resistance. In the hlyD sequence coding for HlyD protein, two mutations were found in positions: 4126646 and 4126679. Both these mutations resulted in deletions and frameshift mutations in the encoded HlyD protein (Table 1). In the case of the tuf gene coding for elongation factor EF-Tu, two mutations were found in positions 1650291 and 1650528, respectively. These mutations resulted in the exchange of amino acids in the encoded proteins from lysine to arginine and from tyrosine to phenylalanine, respectively ( Table 1).

Sequencing of the genomes of
Adsorption of ΦD5 phage to D. solani phage-resistant mutants. The adsorption of ΦD5 to wildtype D. solani cells was fast. Within 5 min, nearly 90% of phage particles had absorbed to the wild-type strain and more than 95% had bound to this host by 20 min (Fig. 1). In contrast, the adsorption of the phage particles to cells of mutants DsR34 and DsR207 was significantly greatly reduced. Only between 1 and 25% of ΦD5 particles had bound to phage-resistant DsR34 and DsR207 cells within 20 min (Fig. 1).

Phenotypes of D. solani phage-resistant mutants.
The phage-resistant mutants were analyzed for changes in their phenotypes that might be essential for their ecological fitness and virulence in natural and agricultural settings [33][34][35] . There were no differences between the mutants and the wild-type strain in most phenotypes examined in this study. Specifically, phage-resistant mutants, like the wild-type strain, could produce pectinases and proteases, forming cavities on the CVP medium, degrading carboxymethylcellulose and polygalacturonic acid. They all were unable to produce siderophores or grow on medium supplemented with 5% NaCl. Similarly, no difference between WT D. solani and phage-resistant mutants DsR34 and DsR207 was observed in the production of secreted enzymes tested with API-ZYM assays. No differences in cell morphology or size diameter were observed in the phage-resistant mutants compared to the IPO 2222 by examination using transmission electron microscopy (TEM) (Fig. 2). Implementing atomic force microscopy (AFM) imaging did not reveal significant differences in the surface morphology of the analyzed D. solani phage-resistant cells with the scanning method applied. The DsR34 and DsR207 mutants were flagellated, and their measured length, width, and height were like the D. solani IPO 2222 wild-type strain (Fig. 3). The phage-resistant D. solani mutants exhibited comparable morphologies and colony size as that of the WT strain. None of the mutants differed significantly in their generation times in either rich (TSB) or minimal (M9 + glucose) media compared to the wild-type strain. The growth rate of the mutants tested at various temperatures (5,8,15,28, and 37 °C) did not differ from that of the WT strain. The growth of the mutants at pH 4.0 and 10.0 was also similar to that of the wild-type strain. Phage-resistant mutants expressed equivalent susceptibility/resistance to all antibiotics tested as the WT strain. While the WT strain could move by swimming and swarming, both phage-resistant mutants were capable of swimming motility but not swarming motility. No significant differences were observed between the phageresistant mutants and the wild-type IPO 2222 strain in most metabolic phenotypes examined. Both DsR34 and DsR207 mutants differed from the wild-type IPO 2222 strain in a total of only 9 features out of 316 such metabolic traits tested using BIOLOG phenotypic microarrays. All these divergent traits (except their resistance to 4% NaCl), were related to carbohydrate metabolism. The phage-resistant mutants both lost the ability to utilize D-cellobiose, D-turanose, and gentiobiose and became susceptible to 4% NaCl. Simultaneously, DsR34 and DsR207 mutants each gained the ability to use N-acetyl-β-D-mannosamine, L-glutamic acid, inulin, D-tagatose, and malonic acid for growth (Supplementary Table 1). Phage-resistant mutants DsR34 and DsR207 exhibited less rapid sedimentation associated with self-aggregation than the wild-type strain ( Supplementary Fig. 4). The SDS-PAGE patterns of the lipopolysaccharides purified from the ΦD5-resistant mutants DsR34 and DsR207 were indistinguishable from each other. However, compared to the LPS purified from the WT strain, the LPSs isolated from DsR34 and DsR207 mutants lacked one faint band of ca. 30 kDa, which was present in the LPS of the WT strain ( Supplementary Fig. 5). www.nature.com/scientificreports/

Proteomics of D. solani phage-resistant mutants.
To gain more insights into the characteristics of the spontaneous ΦD5-resistant mutants, the proteomes of the DsR34, DsR207, and wild-type strain grown in rich (TSB) and minimal (M9 + glucose) media were compared using SWATH-MS analysis. Differential protein expression was determined by comparing the relative abundance of proteins in phage-resistant mutants and the wild-type strain. Fold changes were considered significant with the q-value (adjusted p-value) < 0.05. Spontaneous phage-resistance affected DsR34 and DsR207 proteomes in both media tested. Among the analyzed strains, SWATH-MS identified a total of 492 D. solani proteins in the rich medium and 672 proteins  www.nature.com/scientificreports/ in the minimal medium. In the rich medium, the core proteome of the WT, DsR34, and DsR207 consisted of 385 proteins, whereas 13, 15, and 24 unique proteins were significantly differentially expressed in cells of the WT, DsR34, and DsR207, respectively. Similarly, in the minimal medium (M9 + glucose), the proteome shared between D. solani wild-type strain and phage-resistant mutants comprised 491 proteins, whereas 14, 48, and 30 proteins were uniquely differentially expressed in WT, DsR34, and DsR207, respectively ( Supplementary Fig. 6). The unique proteins (= significantly differentially expressed) were grouped into 10 categories according to their primary functions that include: (1) protein metabolism, (2) carbohydrate metabolism, (3) lipid metabolism, (4) DNA metabolism, (5) transport and signaling, (6) stress and redox processes, (7) metal ion homeostasis, (8) motility and chemotaxis, and (9) co-factors metabolism and (10) others (miscellaneous proteins with unknown function and/or localization) ( Supplementary Fig. 1). The number of proteins in a particular category differed www.nature.com/scientificreports/ between the analyzed strains and the type of medium tested ( Supplementary Fig. 6A,C). However, among the tested strains, most of the differently expressed proteins were represented by the category protein metabolism, and category of others (proteins with unknown function and localization) in both media tested. Proteins related to DNA metabolism, transport and signaling, metal ion homeostasis, and motility and chemotaxis were not more abundant in the WT strain in the rich medium (TSB) relative to that of the phage mutants. Likewise, proteins related to lipid metabolism, metal ion homeostasis and, motility and chemotaxis were not differentially abundant in the WT strain relative to the mutants in a minimal medium (M9 + glucose). The proteins upregulated in DsR34 in the rich medium were linked with carbohydrate metabolism, transport and signaling, motility, and chemotaxis. Contrary, in the minimal medium, proteins associated with protein metabolism, DNA metabolism, transport and signaling and stress and redox processes were upregulated in this mutant. In the case of the DsR207 mutant, in the rich medium, proteins belonging to two categories were significantly upregulated: those involved in protein metabolism and proteins in the category ' others' with unknown functions. In mutant DsR207, proteins associated with carbohydrate and lipid metabolism were upregulated in the minimal medium compared to the other strains ( Supplementary Fig. 8).
Given that the two phage-resistant mutants might be expected to have alternations in their cell surface that resulted in the decreased ΦD5 adsorption, we compared the proteomes of DsR34 and DsR207 mutants and WT strain for differentially expressed proteins associated with bacterial cell surfaces (= envelope, transmembrane transport, and signaling) ( Table 2). Such proteins constituted 40 and 19% of the total unique proteins expressed in rich and minimal medium, respectively for mutant DsR34. In rich media the fraction of envelope-associated proteins among all unique proteins was 12.5%, whereas, in minimal medium, this fraction was 17% for mutant DsR207 ( Supplementary Fig. 6B,D).

Effect of selected surfactants on the viability of phage-resistant D. solani mutants. Since cell
surface features were evidently modified in DsR34 and DsR207 phage-resistant mutants, we assessed the resistance of WT and mutant D. solani cells to surfactants. No differences were found between the phage-resistant mutants DsR34 and DsR207 and D. solani IPO 2222 wild-type strain in response to Triton X-100, Tween 20, Tween 80, Poloxamer 407, and N-lauroyl sarcosine. In contrast, both phage-resistant mutants exhibited reduced growth in the presence of 0.1% EDTA compared with that of the WT strain. On average, the cell concentrations DsR34 and DsR207 after 16 h incubation in the presence of 0.1% EDTA were only about half that of the WT strain ( Supplementary Fig. 7).

Discussion
Our understanding of the mechanisms underlying interactions of SRP bacteria with lytic bacteriophages remains limited 16,27 . This study explored whether there is a direct link between spontaneous phage resistance and virulence of plant pathogenic D. solani IPO 2222. By focusing on virulence and interrogating the spontaneous phageresistant mutants in plant-related environments, we wanted to assess whether resistance to phage infections impacts the ecological success of the pathogen, defined as the ability to survive in the plant surrounding and efficiently colonize and cause symptoms in plant hosts.
Although most ΦD5-resistant D. solani mutants found in this study were unaffected in virulence (> 99%), we identified two mutants, DsR34 and DsR207, that exhibited a significant reduction in their ability to macerate potato tuber tissues. It was perhaps not a surprise that most phage-resistant D. solani mutants retained virulence. It is of utmost importance for a pathogen to remain virulent 36,37 , as losing the ability to infect the host heavily impacts its ability to generate large population sizes thus its survival and ecological success 38 . On the other hand, in complex environments such as the surface and interior of plants, the evolution of resistance to phage infections is likely to be very costly. Phage selective pressure is expected to be a strong driver of spontaneous mutations 39 . In contrast, a direct link between spontaneous resistance to viral infections and reduced virulence has already been shown for members of several bacterial species 40 , including plant pathogenic bacteria, but to our knowledge, not for D. solani. For example, spontaneous resistance to bacteriophage X2 by the in-plant pathogen Xanthomonas oryzae pv. oryzae resulted in reduced virulence 41 . Reduced virulence was also observed in the case of spontaneous resistance of Pseudomonas syringae pv. porri to phages KIL3b and KIL5 42 . It suggests a direct tradeoff cost for phage-resistant mutants in a plant environment. Likewise, recent studies on human pathogens Staphylococcus aureus 43 and Serratia marcescens 44 suggested that selective pressure conferred by the presence of lytic bacteriophages results in the appearance of spontaneous phage-resistant mutants having a lower virulence. Such observations highlight the global link between viral resistance and the ability to cause infections by bacterial pathogens 45 .
Gram-negative bacteria have developed several strategies to cope with viral infections 7 . Among different mechanisms of phage evasion, modification of the bacterial cell surface that prevents phage attachment has been often reported 10 . In this study, the adsorption of ΦD5 to D. solani mutants DsR34 and DsR207 was greatly reduced. This indicates that probably the preferred mechanism of spontaneous resistance against phage ΦD5 in D. solani IPO 2222 is the modification of the cell surface in a way to be unsuitable for a phage attachment (i.e. adsorption inhibition) 11 .
The sequencing of the DsR34 and DsR207 genomes showed that spontaneous ΦD5 resistance was caused by mutations in two distinct loci not connected so far with phage resistance in SRP bacteria 46 : (i) in the gene coding for secretion protein HlyD (mutant DsR34) and (ii) in the gene encoding an elongation factor Tu (EF-Tu) (mutant DsR207). HlyD secretion protein is a conserved trimeric transmembrane fusion protein and a part of the type I secretion system (T1SS) in Gram-negative bacteria. HlyD is involved in the secretion of α-hemolysin in Escherichia coli 47 . In Dickeya spp., a HlyD homolog, protein PrtE takes part in protease secretion 48 , contributing to virulence in planta 49 www.nature.com/scientificreports/ www.nature.com/scientificreports/ proteases during infection 51 . Nevertheless, in our study, the DsR34 mutant maintained the ability to produce proteases. PrtE/HlyD has not yet been previously connected with phage-host interactions, including interactions of SRP members with their bacteriophages. The other mutated protein, elongation factor Tu (EF-Tu), is a G protein catalyzing binding of aminoacyl-tRNA to ribosomes 52 . As such, it has an important role in translation. In addition to its primary function in translation, EF-Tu has the ability to execute diverse tasks on the surface of bacterial cells, including interaction with membrane receptors and the extracellular matrix 52 . In addition, EF-Tu is a recognized pathogen-associated molecular pattern (PAMP) that is recognized by plants 53,54 . In this study, DsR207 mutant has two missense mutations in the EF-Tu gene. It can be speculated that these two mutations may altered the protein in a way that the mutated version is better recognized as a PAMP by the plant than the wild-type EF-Tu 55 , resulting in a strong immune response, thus reducing the DSR207 virulence. EF-Tu is important in the interaction of several plant pathogens, including P. carotovorum, Ralstonia solanacearum, and Agrobacterium tumefaciens, with their host plants acting as an elicitor of the plant innate immune response 56 . Although no reports clearly link EF-Tu with phage-SRP bacteria interaction, it is known that Escherichia coli EF-Tu participates in the bacteriophage exclusion system (altruistic suicide of infected cells upon infection), preventing propagation of T4 phage in the environment 57 . It remains unclear whether Dickeya spp. EF-Tu may work in a similar way as in E. coli.
Spontaneous resistance of DsR34 and DsR207 against ΦD5 had pleiotropic phenotypic consequences. Both mutants identified in this study retain several phenotypic alternations that differentiate them from the WT strain, which may decrease their fitness in planta. Both mutants were unable to swarm, were significantly more susceptible to EDTA, and expressed less rapid cell sedimentation/aggregation compared to the wild-type IPO 2222 strain. Swarming is known to be one of the vital features needed by D. solani to colonize and establish an infection in plants 58 . It is therefore not a surprise that inability to swarm observed in phage-resistant mutants may result in decreased virulence of the DsR34 and DsR207 mutants in planta 38 . Similarly, EDTA is known to increase the permeability of the capsule in Gram-negative bacteria, making them more susceptible to environmental stresses 59 . Similarly, differences in self-aggregation of phage-resistant mutants compared to the wild-type strain suggested changes in the surface features of D. solani cells 60 . Spontaneous mutations conferring phage resistance directly affected the proteomes of DsR34 and DsR207 mutants, including the abundance of the proteins associated with cell envelope in D. solani IPO 2222. Although the two mutations found in this study have not yet been linked to the resistance of SRP or other Gram-negative bacteria to viral infections 7 , they affected the expression of various proteins associated with transmembrane transport, cell wall organization, and metabolism of envelope-associated carbohydrates. Such a phenotype directly links phage resistance with the status of the bacterial envelope, a common feature of phage resistance. Consistent with the conjecture, analysis of the LPS derived from both DsR34 and DsR207 revealed altered LPS. Such a phenotype may explain the decreased attachment of phages to the cells of these mutants. In our earlier work, which analyzed the interaction of P. parmentieri with lytic phage ΦA38, we discovered that this phage required native LPS to infect its host 46 . We showed previously that mutations targeting the LPS cluster in D. solani 61 resulted, among other phenotypes, in resistance to bacteriophage infections 46 . It seems that modifications of LPS and capsule to prevent viral infections frequently occur in bacteria belonging to Soft Rot Pectobacteriaceae, as similar observations were made for other members of this group, including P. atrosepticum, P. carotovorum, and P. brasiliense [62][63][64] . The apparent differences in LPS structure, elevated EDTA susceptibility, and less rapid self-aggregation further strengthened the hypothesis that alternations of bacterial surface properties occurred in phage-resistant mutants DsR34 and DsR207 65 . It is not clear yet how the mutations in HlyD may result in alternations in D. solani surface properties, however, it has been demonstrated that HlyD interacts with the LPS of D. solani 45 . Therefore, mutations of HlyD may result in its incorrect folding, insertion and localization of protein in the periplasm and by these may also affect the LPS structure 49 . Another possibility is that mutations found in HlyD may cause misfolding of the secreted proteins and therefore contributed to the decreased virulence of DsR34 mutant. Point mutations of HlyD were reported to impact the folding of the HlyA protein and its translocation in E. coli 66 . Similar situation may take place in D. solani. The other mutated protein, EF-Tu, was reported to be a part of bacterial cytoskeleton, required to maintain the proper shape of the bacterial cell 67 . EF-Tu localizes underneath the cell membrane of E. coli and Caulobacter crescentus 68 . In Bacillus subtilis, EF-Tu affects cell surface and the decrease in the concentration of EF-Tu leads to defects in cell surface morphology 68 . The interaction of EF-Tu and cytoskeleton elements seems to by an universal mechanism by which the prokaryotic cells sustain their morphological features 69 .
To support the role of D. solani surface alterations in phage avoidance, we used microscopic techniques to compare the surface of the wild-type strain and the spontaneous ΦD5-resistant mutants DsR34 and DsR207. Phage-resistant mutants were overall indistinguishable from the WT strain in the colony and cell morphology. To our surprise, also more detailed analyses done with TEM and AFM did not reveal any apparent alterations in the cell surface of the ΦD5-resistant mutants that could easily explain the observed phenotypes, including the lowered virulence in planta. The DsR34 and DsR207 cells possessed the same surface topography, diameter, and size as the wild-type IPO 2222. It is likely however, that the changes in cell surface LPS and proteins composition would have led to very modest changes in physical appearance of the envelope surface, with such changes being undetectable by even AFM.
The two phage-resistant mutants exhibited reduced virulence in detached chicory leaf assay as well as reduced colonization and symptom expression in potato plants growing in potting soil. Such results indicate that both mutants were heavily compromised in their ability to invade and multiply within different hosts 55 . Their decreased virulence of the phage-resistant mutants after introduction into chicory leaves and in potato plants after soil inoculation was not due to intrinsic reductions in growth rate; under in vitro conditions, the mutants and the WT strain had comparable generation times under a number of conditions, including when grown at different temperatures, pHs, and in the presence of various carbon sources. It is clear, therefore, that the observed ΦD5 resistance strongly impacted both the multiplication of D. solani in planta and thus its ability to generate www.nature.com/scientificreports/ secondary inoculum for persistence and infection of other plants. For this reason, inhibition of phage adsorption by D. solani to avoid infections will likely reduce its ecological fitness in many cases. Such an observation may have practical importance, since implementing phage therapy against such an agricultural pathogen may result not only in the killing of the pathogen but also in the selection of less virulent phage-resistant variants that would reduce the future risk of infection by that species 70 .

Materials and Methods
Bacteriophages, bacterial strains, and growth conditions. The lytic bacteriophage vB_Dsol_D5 (ΦD5), described previously 30,31,71 , was grown on its host, D. solani IPO 2222 21 , and quantified using a soft top agar assay 30 . A stock of phage particles (10 8 -10 9 PFU mL -1 ) in tryptone soya broth (TSB, Oxoid, UK) or quarterstrength Ringer's buffer (Merck, Poland) was used in this study. The wild type (WT) D. solani was cultivated for 24-48 h at 28 °C on tryptone soya agar (TSA, Oxoid, UK), in TSB or M9 medium (MP Biomedicals, the Netherlands) supplemented with 0.4% glucose (Sigma-Aldrich, Poland). To solidify the media, 15 g L -1 bacteriological agar (Oxoid, UK) was added when needed. Spontaneous ΦD5-resistant D. solani mutants were cultivated under the same conditions and using the same growth media as the WT strain. To prevent fungal growth, the growth medium was supplemented with 200 μg mL -1 cycloheximide (Sigma-Aldrich, Poland).

Recovery of spontaneous D. solani phage-resistant mutants.
The recovery of ΦD5-resistant mutants of D. solani was made as previously described 72 . Briefly, the overnight culture of the WT strain grown in TSB (rich medium) or M9 + 0.4% glucose (minimal medium) was diluted 50 times in the same fresh medium. The diluted bacterial culture was grown at 28 ºC with shaking at 120 rpm to achieve 0.5 at OD 600 . Cultures were then spiked with ΦD5 particles suspended in sterile TSB to reach the final MOI of 0.01 following 72-96 h incubation under the same conditions. Bacteria surviving phage infections were purified into individual colonies by at least four passages on TSA agar plates 73 and collected for further studies. Verifying ΦD5 resistance of the obtained D. solani mutants was done as described previously 46 . D. solani IPO 2222 mutants with confirmed ΦD5 resistance were selected for further studies.

Virulence of D. solani phage-resistant mutants.
The virulence of spontaneous phage-resistant mutants was tested initially using a whole potato tuber assay 46 . Briefly, potato tubers (5 replicate tubers per bacterial strain) of cv. Bryza, purchased locally in Gdansk, Poland, were used. They were chosen for their similar size (diameter of 5-6 cm and weight of 50-70 g) 46 and inoculated with a given strain and evaluated for disease symptoms 34,75 . Bacterial suspensions of 10 8 CFU mL −1 , were delivered to the potato tuber by stab inoculation into the tuber pith with a 200 μl pipette tip filled with 100 μl of bacterial suspension. Inoculated tubers were kept in humid boxes (ca. 90% relative humidity) at 28 °C for 72 h to promote the expression of disease symptoms. Positive control tubers were inoculated with the WT D. solani strain, and the negative control tubers were inoculated with sterile demineralized water. The experiment was repeated once. Phage-resistant D. solani mutants demonstrating reduced ability to macerate potato tuber tissues were selected for further analysis.

Identification of genomic alternations in D. solani phage-resistant mutants. DNA of selected
phage-resistant mutants was isolated using a Wizard Genomic DNA purification kit (Promega, Poland) according to the procedures given by the manufacturer. Genome sequencing was done using second and third-generation of next-generation sequencing techniques 76 . For short-read sequencing, samples were quantified and diluted according to service provider specifications and sent offsite for processing. Long-reads (Oxford Nanopore Technologies) sequencing was carried out using Ligation Sequencing Kit (SQK-LSK110) and Native Barcoding Kit 96 (SQK-NBD112.96) according to manufacturers' protocols. Before library preparation, DNA was quantified using the Quantus system (Promega, Poland). The integrity of samples was analyzed with TapeStation 2200 (Agilent, Perlan Technologies, Poland). The library was sequenced using R9.4.1 MinION Flow Cell (FLO-MIN106D) for 72 h 77 . Base-calling and demultiplexing were carried out using the MinKnow software suite using the Super Accuracy option (www. nanop orete flych. com). Contigs were assembled by a hybrid approach where long reads were initially used to generate a whole closed chromosome using the wf-bacterial-genomes workflow from EPI2ME Labs (https:// github. com/ epi2me-labs/ wf-bacte rial-genom es). This workflow uses flye software (https:// github. com/ fende rglass/ Flye) for assembling and medaka software (https:// github. com/ nanop orete ch/ medaka) for long read-based contig polishing. In the next step, contigs were further polished with short reads using three tools: Polypolish (https:// github. com/ rrwick/ Polyp olish), Pilon (https:// github. com/ broad insti tute/ pilon), and POLCA 78  www.nature.com/scientificreports/ the D. solani WT reference genome 32 . Genes found to have changes in their nucleotide sequence were characterized for their transcriptional organization using Operon-mapper (https:// bioco mputo. ibt. unam. mx/ operon_ mapper/) 79 . Analyses of the biochemical pathways in which the selected mutated proteins might participate were done using KEGG 80 . Similarly, mutated proteins were assessed for their possible roles in metabolic and functional cellular networks using STRING (Search Tool for Retrieval of Interacting Genes/Proteins) v11.5 (https:// string-db. org/) (parameters: network type: full network, network edges: high confidence, interaction sources: text mining, experiments, databases, co-expression, co-occurrence, gene fusion 46 ), providing essential information concerning interactions of proteins of interest 81 using the proteome of D. solani IPO 2222 as a reference.
Assessment of ΦD5 adsorption to D. solani phage-resistant mutants. The rate of ΦD5 adsorption to D. solani IPO 2222 WT and phage-resistant mutants was determined as described before 46 . Briefly, bacterial cultures in their log-phase growth were inoculated with a phage suspension (at a Multiplicity of Infection (MOI) of 0.01) and incubated for up to 20 min at 28 °C. Two individual samples of each bacterial strain tested were collected at various times: (0 (control), 1, 2, 5, 10, 15, and 20 min) 46 , and the number of non-adsorbed phages was quantified. Bacteriophages suspended in sterile TSB medium and recovered at the same time points as above were used as a negative control. The experiment was repeated 3 times, and the results were averaged. Phage adsorption efficiency was calculated as described before 46 .
Assessment of bacterial morphology with microscopic techniques. The colony morphology of phage-resistant mutants was analyzed with a Leica MZ10 F stereomicroscope with 10 × and 40 × magnifications coupled to a Leica DFC450C camera as previously described 33,79 . The cell morphology of phage-resistant mutants was evaluated using transmission electron microscopy (TEM) 34 . WT and phage-resistant mutants were adsorbed onto carbon-coated grids (GF Microsystems), directly stained with 1.5% uranyl acetate (Sigma-Aldrich), and visualized with an electron microscope (Tecnai Spirit BioTWIN, FEI) 34 . At least ten images of each ΦD5-resistant bacterial variant and the WT strain were obtained to estimate cell diameter. For atomic force microscopic (AFM) analysis, bacteria were grown overnight in the M9 minimal medium supplemented with 0.4% glucose on a microscopy glass cover slide placed in a Petri dish, with gentle shaking (60 rpm) at 28 °C. The slides were washed with distilled water, and subsequently, samples were fixed with filtered 2.5% glutaraldehyde (Sigma Aldrich) for 2 h, washed again, and air-dried. Cells were imaged using Bioscope Resolve (Bruker), in ScanAsyst (Peak Force Tapping) mode, with the application of ScanAsyst Air probe (f0 7.0 kHz, diameter < 12 nm, k:0.4 N/m) 82 . Post-imaging analysis and cell measurements (n = 12 to 20 cells) were performed using NanoScope Analysis 1.80 (Bruker).

Growth of D. solani phage-resistant mutants .
To determine whether phage resistance affects the growth rate of D. solani mutants, bacterial growth was assessed in both TSB (rich medium) and M9 + 0.4% glucose (minimal medium), as previously described 85 . The experiment was replicated once, and the average generation time was determined using the Doubling Time Calculator (parameters: C0 = 3 h, Ct = 7 h, t = 4 h) (http:// www. doubl ing-time. com/ compu te. php) 86 . In addition, the ability of ΦD5-resistant mutants and the WT to grow at different temperatures was tested qualitatively on solid rich and minimal media incubated at 5, 8, 15, 28, and 37 °C, as described before 86 . Growth was assessed visually on a daily basis. The experiment was repeated once using the same setup. To evaluate whether the ΦD5-resistance affects the growth rate of the mutants at different pHs, the growth rate of selected D. solani ΦD5-resistant mutants was compared in TSB at pH 4 and 10, similarly to other studies 75 . In brief, overnight bacterial cultures in TSB (ca. 10 9 CFU ml -1 ) were diluted 50-fold in fresh growth broth with pH 4 or 10. 100 µL of such prepared bacterial cultures were transferred to the wells of 96-well plates and wrapped with optically clear sealing tape (Sarstedt) to prevent drying up. The bacterial growth rate was determined by optical density (at 600 nm, OD 600 ) measurements every 0.5 h for 16 h in an Epoch2 Microplate Spectrophotometer (BioTek, Poland). The experiment was repeated once, and the results were averaged. The generation time was calculated as described above.

Phenotypes of D. solani phage-resistant mutants.
The ability of phage-resistant D. solani mutants to use different carbon and nitrogen sources was analyzed in GEN III, EcoPlate, PM1, and PM2a 96-well plates in a BIOLOG phenotypic microarray system (Biolog Inc., Biomaxima, Poland) as described previously 79 . In addition, the spontaneous ΦD5-resistant D. solani mutants were also screened for phenotypic features that may be crucial for their environmental fitness 46 , including susceptibility to hydrogen peroxide 75 , swimming and swarming motility 75 , biofilm formation 87 , the capability to grow on TSA medium containing 5% NaCl 88 , production of enzymes: pectinolytic enzymes 89 , cellulases 90 , proteases 91 and siderophores 92 . In addition, to test whether the phage resistance affects the profile of extracellular enzymes produced by D. solani IPO 2222, the phage-resistant mutants were tested, in duplicates, using API-ZYM stripes (bioMérieux, France) following the protocol provided by manufacturer 33 .  Poland). Bacterial cultures in a 96-well plate were shaken (orbital shaker, 60 rpm) between the OD 600 measurements to prevent anaerobic conditions and sedimentation of bacterial cells at the bottom of the well. The growth of each strain was analyzed in 6 technical replicates, and the results were averaged. Six wells inoculated with sterile growth medium served as a negative control, and the positive control was six wells inoculated with bacterial cultures grown in M9 medium + 0.4% glucose. The experiment was repeated once and the results were averaged.  www.nature.com/scientificreports/ above. D. solani wild-type strain IPO 2222 was used as a control. The experiment was repeated once, and the results were averaged.

Autoaggregation of D. solani phage-resistant mutants.
The autoaggregation of phage-resistant D.
solani mutants was measured as enhanced sedimentation and phase separation of aqueous bacterial suspensions, as previously described 98 . Phage-resistant mutants were evaluated for the rate of autoaggregation (sedimentation) as described in 61,99 . Briefly, the WT strain and ΦD5-resistant mutants were grown in 10 ml of TSB at 28 °C with shaking (120 rpm) for 24 h, and 1 ml of bacterial culture was then transferred to sterile cuvettes (Eppendorf). The optical density (OD 600 ) was initially measured (time = 0 h), and after incubation at 28 °C for 24 h. Each phage-resistant bacterial mutant was analyzed in duplicates, the experiment was repeated once, and the results were averaged 57 . Ratio aggregation (sedimentation) was assessed as follows: %A = 1-(OD 600  Virulence of D. solani phage-resistant mutants on chicory leaves. The ability of ΦD5-resistant bacterial mutants to cause maceration of chicory leaves was assessed as previously described 101 . Briefly, chicory leaves (5 replicate leaves per bacterial strain) were purchased locally in Gdansk, Poland. The leaves were inoculated with a given bacterial strain by creating a shallow, ca. 1-cm-long cut across each leaf with a sterile scalpel and subsequently inoculating with 10 µL of bacterial suspension (ca. 10 8 colony forming units (CFU) mL -1 ). Inoculated chicory leaves were sealed in a plastic bag containing filter paper moistened with sterile water. After incubation for 48 h at 28 °C, the diameter of the rotted tissue on each inoculated leaf was measured 101 . WT D. solani was used as a positive control, and negative control tubers were inoculated with sterile demineralized water. The experiment was repeated once, and the results were averaged.

Virulence of D. solani phage-resistant mutants on potato plants. Experiments involving plants
grown in a growth chamber were performed using the previously developed protocol 71 . Certified potato tubers (cv. Kondor) were purchased from the Plant Breeding and Acclimatization Institute (National Research Institute, Poland) and were cultivated as described earlier 71 . After 2 weeks, rooted plants (10-15 cm in height) were transferred to 1 L pots and grown in potting soil for additional 2 weeks. Potato plants (5 plants per repetition) were inoculated with phage-resistant D. solani mutants or the WT strain by the application of 50 ml of bacterial suspensions (10 8 CFU mL −1 ) in sterile Ringer's buffer directly to the soil surrounding the base of stems 1 h after plants had been well irrigated to guarantee uniformly moist soil. As a negative control, the soil was treated with sterile Ringer's buffer (50 ml per plant) alone. Pots were then randomized in a growth chamber with 4 blocks of 10 pots. Plants were visually examined daily for the development of disease symptoms (chlorosis, black rotting of the stem, haulm wilting, plant death). Plants were sampled for bacterial populations 14 days after inoculation. Per the analyzed plant, stem sections (approx. 2 cm long) located approx. 5 cm above ground level were collected, combined into one sample, and surface-sterilized as described before 79 . The number of bacterial cells within potato stems was determined by dilution-plating stem macerates on CVP medium supplemented with 200 μg mL −1 cycloheximide and counting the resulting cavity-forming D. solani colonies. The experiment was repeated once, and the results were averaged for analysis.

Statistical analysis.
Statistical analyses were carried out using a previously described approach 46 . To achieve normality, bacterial colony counts were transformed to log 10 (x + 1) 102 . Treatments were examined following the experimental design, which, each time, involved doing two independent duplicated experiments for every treatment applied. For samples in which bacterial population sizes were normally distributed, the Shapiro-Wilk test (at p = 0.05) was applied 103 . The Welch's T-test was used for samples in which population sizes were not normally distributed, such as: (i) control vs. treatment or (ii) treatment vs. treatment 104 . The Fisher-Snedecor test was used to validate the homogeneity of variance 105 . A two-tailed Student's t test was used to assess pairwise differences between analyzed samples 106 . The linear model included an entire block design, and replicates were treated as separate blocks 107 . The impact of time, the treatment, and the two-way interaction between the time and treatment type were examined in the model.

Data availability
Data generated or analyzed during this study are included in this published article and its supplementary materials. Correspondence and requests for materials should be addressed to R.C. In addition, the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD038825. The DsR34 and DsR207 complete genomes were deposited in the NCBI www.nature.com/scientificreports/